The present invention is directed to the use of recombinant DNA techniques to confer upon microorganism host cells the capacity for selected bioconversions. More specifically, the invention is directed to the cloning of toluene monooxygenase genes from a newly isolated and characterized Pseudomonas strain, Pseudomonas mendocina KR-1. The present invention provides genetically engineered plasmids that allow production of toluene monooxygenase enzymes and proteins in a variety of Gram-negative bacteria in the absence of a toxic inducer, and provides more efficient means of conducting bioconversions dependent on this enzyme system.
A bacterial strain identified as Pseudomonas mendocina KR-1 (PmKR1) was isolated by Richardson and Gibson from an algal-mat taken from a fresh water lake. Whited, Ph.D. Dissertation, The University of Texas at Austin, Library Reference No. W586 (1986). PmKR1 utilizes toluene as a sole carbon and energy source. Other bacterial strains have been isolated and described which metabolize or degrade toluene, including Pseudomonas putida mt-2 (Pp mt-2) (Williams and Murray, J. Bacteriol. 120: 416-423 (1974) and Pseudomonas putida PpF1 (PpF1) (Gibson, et al. Biochemistry 9:1626-1630 (1970)). In addition, a bacterial strain designated G4, isolated from a waste treatment lagoon, can metabolize toluene (Shields et al., App. Environ. Microbiol. 55: 1624-1629 (1989)). However, the genes, the enzymes and the pathways for toluene metabolism in these various bacterial strains are distinct and non-overlapping.
The catabolic pathway for the degradation of toluene by Pp mt-2 has been designated TOL. The genes for the TOL pathway are encoded on isofunctional catabolic plasmids found in certain strains of Pseudomonas. The reference plasmid for the TOL degradative pathway is pWWO originally isolated from Pp mt-2. The genetics and biochemistry of the TOL pathway are well described. Kunz and Chapman, J. Bacteriol. 146:179-191 (1981); Williams and Murray, J. Bacteriol. 120:416-423 (1974); Williams and Worsey, J. Bacteriol. 125:818-828 (1976); Worsey and Williams, J. Bacteriol. 124:7-13 (1975); Murray, et al., Eur. J. Biochem. 28:301-310 (1972). In particular, detailed studies of the organization and regulation of the TOL pathway genes of plasmid pWWO have been performed. Franklin, et al., Proc. Natl. Acad. Sci. U.S.A. 78: 7458-62 (1981); Spooner et al., J. Gen. Microbiol. 132: 1347-58 (1986); Spooner, et al., J. Bacteriol. 169:3581-86 (1987); Inouye et al., J. Bacteriol. 169: 3587-92 (1987); Inouye et al., Gene 66: 301-306 (1988). A brief summary of the TOL pathway is as follows: initial attack of toluene is at the methyl group which undergoes successive oxidations to form benzoic acid, which is further oxidized by formation of a cis-carboxylic acid diol, which is oxidized to form catechol, which is then degraded by enzymes of a meta cleavage pathway to acetaldehyde and pyruvate.
A second catabolic pathway for the degradation of toluene by PpF1 has been established and designated TOD. In contrast to the TOL pathway, the genes for the TOD pathway are located on the bacterial chromosome and are not plasmid-encoded. Finette, et al., J. Bacteriol. 160:1003-1009 (1984); Finette, Ph.D. Dissertation, The University of Texas at Austin, Library Reference No. F494 (1984). The genetics and biochemistry of the TOD pathway has been studied by Finette, et al. (supra); Finette (Supra); Gibson, et al. Biochemistry 9:1626-1630 (1970); Kobal, et al., J. Am. Chem. 95:4420-4421 (1973); Ziffer, et al., J. Am. Chem. Soc. 95:4048-4049 (1973); Dagley, et al., Nature 202:775-778 (1964); Gibson, et al., Biochemistry 7:2653-2662 (1968). A brief summary of the TOD pathway is as follows: the initial attack of toluene is by a dioxygenase enzyme system to form (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene(cis-toluene dihydr odiol) which is oxidized to 3-methylcatechol which is further degraded by enzymes of a meta cleavage pathway. Zylstra and Gibson, J. Biol. Chem. 264: 14940-46 (1989) and McCombie, Abstr. Annu. Meet. Am. Soc. Microbiol. K53: 155 (1984) have reported the cloning and sequencing of the cod genes which encode the first three enzymes in the TOD pathway.
A third catabolic pathway for the degradation of toluene has been recently identified in PmKR1. It has been found that PmKR1 catabolizes toluene by a novel pathway which is completely different than either of the two pathways described above. Richardson and Gibson, Abstr. Annu. Meet. Am. Soc. Microbiol. K54:156 (1984). The catabolic pathway for the degradation of toluene by PmKR1 has been designated TMO, because the first step in the pathway is catalyzed by a unique enzyme complex, toluene monooxygenase. The biochemistry of the partially purified enzymes and proteins of this pathway has been recently studied by Whited, Ph.D. Dissertation, The University of Texas at Austin, Library Reference No. W586 (1986).
More recently, a toluene catabolic pathway, apparently distinct from the three above-described pathways, has been described in the trichloroethylene-degrading bacterium G4 by Shields et al., App. Environ. Microbiol. 55:1624-1629 (1989). The bacterial strain designated G4 was isolated from a waste treatment lagoon. Strain G4 is uncharacterized with respect to genus and species. The toluene pathway of G4 appears to involve dihydroxylations of the aromatic ring by two monooxygenations, first ortho and then meta. The enzymes involved in these reactions have not been isolated and studied, and therefore remain completely uncharacterized.
The steps of the TMO pathway as outlined by Whited (supra) are diagrammed in FIG. 1. In the initial step toluene is oxidized to p-cresol, followed by methyl group oxidation to form p-hydroxybenzoate, followed by hydroxylation to protocatechuate and subsequent ortho ring cleavage. In the first step of the TMO pathway, toluene is converted by toluene monooxygenase to p-cresol. PmKR1 elaborates a unique multicomponent enzyme system which catalyzes this first step monooxygenase reaction. The implications of the teachings of Whited, (supra), suggest that at least three protein components may be involved: component a (possibly NADH oxidoreductase, molecular weight unknown), component b (possibly an oxygenase, at least 2 subunits) and component c (red-brown, probably ferredoxin, 23,000d.).
Despite beginning biochemical studies of the enzymes and proteins of the TMO pathway (Whited, supra) and beginning genetic studies (Yen et al. Abstract, University of Geneva EMBO Workshop, Aug. 31-Sep. 4, 1986), the art has not been provided with information regarding the genes encoding the enzymes and proteins of the toluene monooxygenase system in PmKR1 or the usefulness of such genes and gene products in certain microbial bioconversions. The art has also not been provided with microorganism host cells harboring novel recombinant plasmids containing PmKR1 toluene monooxygenase genes, in which induction of the toluene monooxygenase genes does not involve use of toxic compounds or simultaneous induction of other undesirable genes and in which some of the microorganism host cells harboring such recombinant plasmids under certain conditions express toluene monooxygenase enzyme activity at levels that equal, or under certain assay conditions, exceed the activity of wildtype PmKR1 cells.